Eveline Fonzé

Comparison of the sequences of class A beta-lactamases and of the secondary structure elements of penicillin-recognizing proteins.

The sequences of class A beta-lactamases were compared. Four main groups of enzymes were distinguished: those from the gram-negative organisms and bacilli and two distinct groups of Streptomyces spp. The Staphylococcus aureus PC1 enzyme, although somewhat closer to the enzyme from the Bacillus group, did not belong to any of the groups of beta-lactamases. The similarities between the secondary structure elements of these enzymes and those of the class C beta-lactamases and of the Streptomyces sp. strain R61 DD-peptidase were also analyzed and tentatively extended to the class D beta-lactamases. A unified nomenclature of secondary structure elements is proposed for all the penicillin-recognizing enzymes.

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.

Abstract: Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of β-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for β-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 Å. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451–465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for β-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.

TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant.

beta-Lactamases are bacterial enzymes which catalyse the hydrolysis of the beta-lactam ring of penicillins, cephalosporins and related compounds, thus inactivating these antibiotics. The crystal structure of the TEM1 beta-lactamase has been determined at 1.9 A resolution by the molecular-replacement method, using the atomic coordinates of two homologous beta-lactamase refined structures which show about 36% strict identity in their amino-acid sequences and 1.96 A r.m.s. deviation between equivalent Calpha atoms. The TEM1 enzyme crystallizes in space group P2(1)2(1)2(1) and there is one molecule per asymmetric unit. The structure was refined by simulated annealing to an R-factor of 15.6% for 15 086 reflections with I >/= 2sigma(I) in the resolution range 5.0-1.9 A. The final crystallographic structure contains 263 amino-acid residues, one sulfate anion in the catalytic cleft and 135 water molecules per asymmetric unit. The folding is very similar to that of the other known class A beta-lactamases. It consists of two domains, the first is formed by a five-stranded beta-sheet covered by three alpha-helices on one face and one alpha-helix on the other, the second domain contains mainly alpha-helices. The catalytic cleft is located at the interface between the two domains. We also report the crystallographic study of the TEM S235A mutant. This mutation of an active-site residue specifically decreases the acylation rate of cephalosporins. This TEM S235A mutant crystallizes under the same conditions as the wild-type protein and its structure was refined at 2.0 A resolution with an R value of 17.6%. The major modification is the appearance of a water molecule near the mutated residue, which is incompatible with the OG 235 present in the wild-type enzyme, and causes very small perturbations in the interaction network in the active site.

The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of streptomyces K15.

The serinedd-transpeptidase/penicillin-binding protein ofStreptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. TheStreptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, theStreptomyces K15 dd-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of theStreptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-Å resolution and refined to anR-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A β-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal α-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded β-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A β-lactamases, except that the Ω-loop, which bears the essential catalytic Glu166residue in the class A β-lactamases, is entirely modified. This loop adopts a conformation similar to those found in theStreptomyces R61 dd-carboxypeptidase and class C β-lactamases, with no equivalent acidic residue.

X-RAY STUDIES OF ENZYMES THAT INTERACT WITH PENICILLINS

Abstract. The technique of X-ray diffraction has been successfully applied to enzymes associated with peptidoglycan biosynthesis. The technique has taught us a great deal about the structures and catalytic mechanisms of penicillin-binding proteins and β-lactamases. An insight into the structural basis for antibiotic resistance is given.

Crystal Structure of the Mycobacterium Fortuitum Class a {Beta}-Lactamase: Structural Basis for Broad Substrate Specificity.

ABSTRACT β-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A β-lactamases, the largest group of β-lactamases, have been found in many bacterial strains, including mycobacteria, for which no β-lactamase structure has been previously reported. The crystal structure of the class A β-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-Å resolution. The enzyme is a chromosomally encoded broad-spectrum β-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A β-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum β-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum β-lactamases a more efficient cefotaximase activity.

Structural basis of the inhibition of class A beta-lactamases and penicillin-binding proteins by 6-beta-iodopenicillanate

6-Beta-halogenopenicillanates are powerful, irreversible inhibitors of various beta-lactamases and penicillin-binding proteins. Upon acylation of these enzymes, the inhibitors are thought to undergo a structural rearrangement associated with the departure of the iodide and formation of a dihydrothiazine ring, but, to date, no structural evidence has proven this. 6-Beta-iodopenicillanic acid (BIP) is shown here to be an active antibiotic against various bacterial strains and an effective inhibitor of the class A beta-lactamase of Bacillus subtilis BS3 (BS3) and the D,D-peptidase of Actinomadura R39 (R39). Crystals of BS3 and of R39 were soaked with a solution of BIP and their structures solved at 1.65 and 2.2 A, respectively. The beta-lactam and the thiazolidine rings of BIP are indeed found to be fused into a dihydrothiazine ring that can adopt two stable conformations at these active sites. The rearranged BIP is observed in one conformation in the BS3 active site and in two monomers of the asymmetric unit of R39, and is observed in the other conformation in the other two monomers of the asymmetric unit of R39. The BS3 structure reveals a new mode of carboxylate interaction with a class A beta-lactamase active site that should be of interest in future inhibitor design.

Crystal structure of Enterobacter cloacae 908R class C beta-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue.

The structures of the class C β-lactamase from Enterobacter cloacae 908R alone and in complex with a boronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 Å, respectively. The structure of the enzyme resembles those of other class C β-lactamases. The structure of the complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C β-lactamase in complex with a transition-state analogue provides further insights into the mechanism of action of these hydrolases.

Expression, purification, crystallization and preliminary X-ray analysis of the native class C beta-lactamase from Enterobacter cloacae 908R and two mutants.

Crystals have been obtained of the Enterobacter cloacae 908R beta-lactamase and two point mutants by the vapour-diffusion method using similar conditions [pH 9.0, polyethylene glycol (M(r) = 6000) as precipitant]. The three crystal forms belong to the orthorhombic space group P2(1)2(1)2, with roughly the same unit-cell parameters; i.e. for the wild-type crystals a = 46.46, b = 82.96, c = 95.31 A. In the best cases, the crystals diffract to about 2.1 A resolution on a rotating-anode X-ray source at room temperature. Co-crystallization experiments of poor substrates with the wild-type protein and the active-site serine mutant (S64C) are planned and should lead to a better understanding of the catalytic mechanism of class C beta-lactamases.